1. Field of the Invention
The present invention is directed to various assays for detection of Axcex2 amyloid, screening candidate agents for their ability to prevent or reverse the formation of Axcex2 amyloid in vitro, as well as kits which are used in the present methods.
2. Related Art
Aggregation of Axcex2 in the brain is believed to contribute to dementia, characteristic of Alzheimer""s disease (AD) and Down""s syndrome, a condition characterized by premature AD. Axcex2, a 4.3-kDa peptide, is the principal constituent of the cerebral amyloid deposits, a pathological hallmark of Alzheimer""s disease (AD) (Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985); Glenner and Wong, Biochem. Biophys. Rev. Commun. 120:885-890 (1984)). Axcex2 is derived from the much larger amyloid protein precursor (APP) (Kang et al., Nature 325:733-736 (1987); Tanzi et al., Science 235:880-884 (1987); Robakis et al., Proc. Natl. Acad. Sci. USA 84:4190-4194 (1987); Goldgaber et al., Science 235:877-880 (1987)), whose physiological function remains unclear. The cause of Alzheimer""s disease remains elusive; however, the discovery of mutations of APP close to or within the Axcex2 domain (Goate et al., Nature 349:704-706 (1991); Levy et al., Science 248:1124-1126 (1990); Murrell et al., Science 254:97-99 (1991); Hendricks et al., Nature Genet. 1:218-221 (1992), linked to familial AD (E. Levy et al., Science 248:1124 (1990); Axcex2 Goate et al., Nature 349:704 (1991); M. Chartier-Harlin et al., Nature 353:844 (1991); J. Murrell, M. Farlow, B. Ghetti, M. D. Benson, Science 254:97 (1991); L. Hendricks et al., Nature Genet. 1:218 (1992); M. Mullan et al., Nature Genet. 1:345 (1992)), indicates that the metabolism of Axcex2 and APP is likely to be intimately involved with the pathophysiology of this disorder.
Soluble Axcex2 is secreted in cell cultures and is found as a 40-residue peptide (Axcex21-40) in the cerebrospinal fluid (CSF) (Shoji et al., Science 258 126-129 (1992); Seubert et al., Nature 359:325-327 (1992); Haass et al., Nature 359:322-325 (1992)), but is not found at elevated levels in sporadic AD cases (M. Shoji et al., Science 258:126 (1992); P. Seubert et al., Nature 359:325 (1992)). Physiological factors which can induce the aggregation of soluble Axcex2 are of interest in determining the cause of Axcex2 amyloid formation. Synthetic Axcex21-40 remains soluble at concentrations up to 16 mg/ml in neutral phosphate buffer (Tomski and Murphy, Arch. Biochem. Biophys. 294:630-638 (1992)), indicating that overproduction of soluble Axcex2 cannot sufficiently explain Axcex2 precipitation. Hence, biochemical mechanisms which promote Axcex2 amyloid formation in sporadic cases would appear to be relevant to the pathogenesis of AD. Furthermore, soluble Axcex2 in cerebrospinal fluid is not increased in AD cases (Shoji et al., Science 258: 126-129 (1992)), indicating that other pathogenetic mechanisms are likely to be involved.
In recent years, the study of Axcex2 peptide has led to making cell lines that express or overexpress Axcex2 or its precursor protein, APP or increased amounts of its more amyloidogenic Axcex21-42 form. See N. Suzuki et al., Science 264:1336-1340 (1994); X-D Cai et al., Science 259:514-516 (1993); F. S. Esch et al., Science 248:1122-1124 (1990). Moreover, monoclonal antibodies to Axcex2 peptide have been generated (see, e.g. U.S. patent Ser. No. 5,231,000, issued Jul. 27, 1993). These monoclonal antibodies are useful as reagents for use in detecting presence of Axcex2 amyloid.
The process described in this invention involves the rapid induction of Axcex2 amyloid by a heavy metal cation such as zinc to form amyloid. In a preferred embodiment of the invention, the proportion of an Axcex21-40 solution which remains filtrable after incubation with zinc is assayed and the effects of candidate pharmacological agents on the filtrate are measured to determine their ability to maintain the solubility of Axcex2 in physiological solution and thus prevent Axcex2 amyloid formation.
A method for the in vitro induction of Axcex2 amyloid has been previously described (J. T. Jarrett et al., Biochem. 32:4693-4697 (1993)). However, this method has many disadvantages, such as a requirement for high concentrations of peptide and prolonged incubation periods (days) with results that are qualitative rather than quantitative. In contrast, some of the major advantages of the present invention are that the technique is reliable, rapid (can be carried out in minutes), is easily quantifiable, and is achieved with low micromolar concentrations of peptide.
Hence, the present invention relates to an in vitro method for the rapid screening of candidate reagents which are likely to be effective in preventing or reversing the formation of amyloid deposits in vivo which are characteristic of Alzheimer""s disease and related pathological conditions. Promising candidate reagents which are selected through one of the in vitro methods of the present invention may then be tested for their effectiveness in vivo in patients which are suffering from Alzheimer""s disease or who are at risk for developing Alzheimer""s disease.
One aspect of the invention relates to a rapid analytical method for detection of Axcex2 amyloid formation in a biological fluid which comprises:
(a) preparing a first set of reaction mixtures comprising neat biological fluid from a control human subject, and serial dilutions of the same made in aqueous buffer or physiological solution;
(b) preparing a second set of reaction mixtures comprising neat biological fluid from a human patient suspected of amyloidosis, and serial dilutions of the same made in aqueous buffer or physiological solution;
(c) adding an equal amount of AB peptide comprising at least amino acids 6 to 28 of Axcex2 to each serial dilution sample;
(d) contacting each of the first and the second set of reaction mixtures with an amount greater than 300 nM of a heavy metal cation capable of binding to an Axcex2 peptide comprising at least amino acids 6 to 28 of Axcex2;
(e) centrifuging each of the first and the second sets of reaction mixtures to give a first and a second set of pellets, respectively; and
(f) comparing the amount of amyloid in the first and the second set of pellets and thereby detecting excessive Axcex2 amyloid formation in the biological fluid from the human patient suspected of amyloidosis.
A second aspect of the invention relates to a method for determining whether a compound inhibits the formation of Axcex2 amyloid which comprises:
(a) pre-filtering an aqueous buffer solution of Axcex2 peptide, which comprises at least the region in the Axcex2 peptide from amino acid number 6 to 28 to give a first filtrate;
(b) measuring the amount of Axcex2 peptide in the first filtrate obtained in step (a);
(c) contacting the first filtrate obtained in step (a) with a heavy metal cation capable of binding to the peptide comprising at least amino acids 6 to 28 of Axcex2 to give a reaction mixture;
(d) contacting the reaction mixture obtained in step (c) with a candidate anti-amyloidotic agent;
(e) filtering the reaction mixture obtained in step (d) to give a second filtrate; and
(f) comparing the amount of Axcex2 peptide in the second filtrate with the amount of Axcex2 peptide in the first filtrate, thereby determining whether the candidate compound inhibits formation of Axcex2 amyloid.
A third aspect of the invention relates to a method for determining whether a compound inhibits formation of Axcex2 amyloid which comprises:
(a) assembling a first and a second reaction mixture, wherein each reaction mixture comprises an equal amount of a pre-filtered Axcex2 peptide solution, which comprises at least the region in the Axcex2 peptide from amino acid number 6 to 28, and an aqueous buffer or physiological solution;
(b) contacting each of the first and the second reaction mixtures with an equal amount of a candidate anti-amyloidotic agent;
(c) contacting the first reaction mixture with a heavy metal cation capable of binding to the peptide comprising at least amino acids 6 to 28 of Axcex2;
(d) contacting the second reaction mixture with EDTA; and
(e) comparing the amount of amyloid formed in the first reaction mixture with that in the second reaction mixture, thereby determining whether the candidate compound inhibits the formation of Axcex2 amyloid.
A fourth aspect of the invention relates to a method for determining whether a compound inhibits formation of Axcex2 amyloid which comprises:
(a) assembling a first and a second reaction mixture, wherein each reaction mixture comprises an equal amount of a prefiltered Axcex2 peptide solution, which contains at least the region in the Axcex2 peptide from amino acid number 6 to 28, and an aqueous buffer or physiological solution;
(b) contacting each of the first and the second reaction mixtures with an equal amount of a candidate anti-amyloidotic agent;
(c) contacting only the first reaction mixture with a heavy metal cation capable of binding to the peptide comprising at least amino acids 6 to 28 of Axcex2; and
(d) comparing the amount of amyloid formed in the first reaction mixture with that in the second reaction mixture, thereby determining whether the compound inhibits formation of Axcex2 amyloid.
A fifth aspect of the invention relates to a method for determining whether a compound inhibits formation of Axcex2 amyloid which comprises:
(a) establishing a first and a second cell culture comprising a cell line which expresses at least a human Axcex2 peptide comprising at least the region of the Axcex2 peptide from amino acid number 6 to 28;
(b) contacting equal concentrations of zinc to each cell culture;
(c) contacting the first cell culture with the candidate agent, and contacting the second cell culture with a heavy metal chealating agent; and
(d) comparing the amount of amyloid and zinc-induced Axcex2 aggregates in each cell culture, thereby determining effectiveness of the candidate anti-amyloidotic agent.
A sixth aspect of the invention relates to a method for determining whether a compound inhibits formation of Axcex2 amyloid which comprises:
(a) establishing a first and a second cell culture comprising a cell line which expresses at least a human Axcex2 peptide comprising at least the region of the Axcex2 peptide from amino acid number 6 to 28;
(b) contacting the first cell culture with zinc to give a first reaction mixture;
(c) contacting the first reaction mixture and the second cell culture with the candidate agent; and
(d) comparing the amount of amyloid and zinc-induced Axcex2 aggregates in each cell culture, thereby determining effectiveness of the candidate anti-amyloidotic agent.
A seventh aspect of the invention relates to a kit for determining whether a compound inhibits formation of Axcex2 amyloid which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein
(a) the first container means contains a peptide comprising at least the region of the Axcex2 peptide from amino acid number 6 to 28; and
(b) a second container means contains a heavy metal cation.
FIGS. 1a, 1b, 1c, 1d and 1e. Analyses of 65Zn2+ binding to Axcex2. Values shown are meansxc2x1S.D., nxe2x89xa73. (1a) Scatchard plot. Aliquots of Axcex2 were incubated (60 min) with 65Zn2+ in the presence of varying concentrations of unlabeled Zn2+ (0.01-50 xcexcM total). The proportion of 65Zn2+ binding to immobilized peptide (1.0 nmol) described two binding curves as shown. The high-affinity binding curve has been corrected by subtracting the low-affinity component, and the low-affinity curve has had the high-affinity component subtracted. (1b) Bar graph showing the specificity of the Zn2+ binding site for metals. Axcex2 was incubated (60 min) with 65Zn2+ (157 nM, 138,000 cpm) and competing unlabeled metal ions (50 xcexcM total). (1c) Bar graph showing 65Zn2+ (74 nM, 104,000 cpm) binding to negative (aprotinin, insulin a-chain, reverse peptide 40-1) and positive (bovine serum albumin (BSA)) control proteins and Axcex2 fragments (identified by their residue numbers within the Axcex2 sequence, gln11 refers to Axcex21-28 where residue 11 is glutamine). Percent binding of total counts 65Zn2+/min added is corrected for the amounts (in nanomoles) of peptides adhering to the membrane. (1d) Scatchard plot. As for (1a), with Axcex21-28 peptide substituting for Axcex21-40. 157 nM 65Zn (138,000 cpm) is used in this experiment to probe immobilized peptide (1.6 nmol). (1e) Graph showing the pH dependence of 65Zn2+ binding to Axcex21-40.
FIGS. 2a, 2b and 2c. Effect of Zn2+ and other metals on Axcex2 polymerization using G50 gel filtration chromatography. Results shown are indicative of n greater than 3 experiments where 55 xcexcg of Axcex2 is applied to the column and eluted in 15 ml, monitored by 254 nm absorbance. (2a) A graph showing the chromatogram of Axcex2 in the presence of EDTA, 50 xcexcM, Zn2+, 0.4 xcexcM; Zn2+, 25 xcexcM; and Cu2+, 25 xcexcM. The elution points of molecular mass standards and relative assignments of Axcex2 peak elutions are indicated. Mass standards were blue dextran (2xc3x97106 kDa, V0=void volume), BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa), and aprotinin (6.5 kDa). The mass of Axcex2 is 4.3 kDa. (2b) Bar graph showing the relative amounts (estimated from areas under the curve) of soluble Axcex2 eluted as monomer, dimer, or polymer in the presence of various metal ions (25 xcexcM), varying concentrations of Zn2+ or Cu2+ (the likelihood of Tris chelation is indicated by upper limit estimates), and EDTA; Data for experiments performed in the presence of copper were taken from 214 nm readings and corrected for comparison. (2c) Bar graph showing the effects of pre-blocking the chromatography column with BSA upon the recovery of Axcex2 species in the presence of zinc (25 xcexcM), copper (25 xcexcM), or chelator.
FIGS. 3a and 3b. Axcex2 binding to kaolin (aluminum silicate): effects of zinc (25 xcexcM), copper (25 xcexcM), and EDTA (50 xcexcM). (3a) Bar graph showing the concentration (by 214 nm absorbance) of Axcex2 remaining in supernatant after incubation with 10 mg of G50 Sephadex. (3b) Bar graph showing the concentration (by 214 nm absorbance) of Axcex2 remaining in supernatant after incubation with 10 mg of kaolin, expressed as percent of the starting absorbance.
FIGS. 4a and 4b. Effect of Zn2+ upon Axcex2 resistance to tryptic digestion. (4a) A blot of tryptic digests of Axcex2 (13.9 xcexcg) after incubation with increasing concentrations of zinc (lane labels, in micromolar), stained by Coomassie Blue. Digestion products of 3.6 kDa (Axcex26-40), and 2.1 kDa (Axcex217-40), as well as undigested Axcex21-40 (4.3 kDa), are indicated on the left. The migration of the low molecular size markers (STD) are indicated (in kilodaltons) on the right. (4b) A bar graph showing 65Zn2+ binding to Axcex2 tryptic digestion products. The blot in a was incubated with 65Zn2+, the visible bands excised, and the bound counts for each band determined. These data are typical of n=3 replicated experiments.
FIG. 5. Scatchard analysis of 65Zn binding to rat Axcex21-40. Dissolved peptides (1.2 nMol) were dot-blotted onto 0.20xcexc PVDF membrane (Pierce) and competition analysis performed as described in Example 1 (FIG. 1). Rat Axcex21-40 and human Axcex21-40 were synthesized by solid-phase Fmoc chemistry. Purification by reverse-phase HPLC and amino acid sequencing confirmed the synthesis. The regression line indicates a KA of 3.8 xcexcM. Stoichiometry of binding is 1:1. Although the data points for the Scatchard curve are slightly suggestive of a biphasic curve, a biphasic iteration yields association constants of 2 and 9 xcexcM, which does not justify an interpretation of physiologically separate binding sites.
FIGS. 6a, 6b, 6c and 6d. Effect of zinc upon human, 125I-human and rat Axcex21-40 aggregation into  greater than 0.2xcexc particles. Stock human and rat Axcex21-40 peptide solutions (16 xcexcM) in water were pre-filtered (Spin-X, Costar, 0.2xcexc cellulose acetate, 700 g), brought to 100 mM NaCl, 20 mM Tris-HCl, pH 7.4 (buffer 1) xc2x1EDTA (50 xcexcM) or metal chloride salts, incubated (30 minutes, 37xc2x0 C.) and then filtered again (700 g, 4 minutes). The fraction of the Axcex21-40 in the filtrate was calculated by the ratio of the filtrate OD214 (the response of the OD214, titrated against human and rat Axcex21-40 concentrations (up to 20 xcexcM in the buffers used in these experiments), was determined to be linear) relative to the OD214 of the unfiltered sample. All data points are in triplicate, unless indicated. (6a) A graph showing the proportions of Axcex21-40, incubated xc2x1Zn2+ (25 xcexcM) or EDTA (50 xcexcM) and then filtered through 0.2xcexc, titrated against peptide concentration. (6b) A graph showing the proportion of Axcex21-40 (1.6 xcexcM) filtered through 0.2xcexc, titrated against Zn2+ concentration. 125I-human Axcex21-40 (125I-human Axcex21-40 was prepared according to the method in Mantyh et al., J. Neurochem 61:1171 (1993) (15,000 CPM, the kind gift of Dr. John Maggio, Harvard Medical School) was added to unlabeled Axcex21-40 (1.6 xcexcM) as a tracer, incubated and filtered as described above. The CPM in the filtrate and retained on the excised filter were measured by a xcex3-counter. (6c) A bar graph showing the proportion of Axcex21-40 (1.6 xcexcM) filtered through 0.2xcexc it following incubation with various metal ions (3 xcexcM). The atomic number of the metal species is indicated. (6d) A graph showing the effects of Zn2+ (25 xcexcM) or EDTA (50 xcexcM) upon kinetics of human Axcex21-40 aggregation measured by 0.2xcexc filtration. Data points are in duplicate.
FIGS. 7a, 7b, 7c and 7d. Size estimation of zinc-induced Axcex2 aggregates. (7a and 7b) Bar graphs showing the proportion of Axcex21-40 (1.6 xcexcM in 100 mM NaCl, 20 mM Tris-HCl, pH 7.4 (buffer 1), incubated xc2x1Zn2+ (25 xcexcM) or EDTA (50 xcexcM) and then filtered through filters of indicated pore sizes (Durapore filters (Ultrafree-MC, Millipore) were used for this study, hence there is a slight discrepancy between the values obtained with the 0.22xcexc filters in this study compared to values obtained in FIG. 6 using 0.2xcexc Costar filters). (7c) A bar graph showing 65ZnCl2 (130,000 CPM, 74 nM) used as a tracer of the assembly of the zinc-induced aggregates of human Axcex21-40 produced in FIGS. 7a and 7b. By determining the amounts of Axcex21-40 and 65Zn in the filtrate, the quantities retarded by the filters could be determined, and the stoichiometry of the zinc: Axcex2 assemblies estimated. (7d) Bar graph. Following this procedure, the filters, retaining Zn: Axcex2 assemblies, were washed with buffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH 7.4)+EDTA (50 xcexcMxc3x97300 xcexcl, 700 g, 4 minutes). The amounts of zinc-precipitated Axcex21-40 resolubilized in the filtrate fraction were determined by OD214, and expressed as a percentage of the amount originally retained by the respective filters. 65Zn released into the filtrate was measured by xcex3-counting.
FIGS. 8a and 8b. Zinc-induced tinctorial amyloid formation. (8a) Zinc-induced human Axcex21-40 precipitate stained with Congo Red. The particle diameter is 40xcexc. Axcex21-40 (200 xcexclxc3x9725 xcexcM in buffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH 7.4)) was incubated (30 minutes, 37xc2x0 C.) in the presence of 25 xcexcM Zn2+. The mixture was then centrifuged (16,000 gxc3x9715 minutes), the pellet washed in buffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH 7.4)+EDTA (50 xcexcM), pelleted again and resuspended in Congo Red (1% in 50% ethanol, 5 minutes). Unbound dye was removed, the pellet washed with buffer 1 (100 mM NaCl, 20 mM Tris-HCl, pH 7.4) and mounted for microscopy. (8b) The same aggregate visualized under polarized light, manifesting green birefringence. The experiment was repeated with EDTA (50 xcexcM) substituted for Zn2+ and yielded no visible material.
FIG. 9. A graph showing the effect of zinc and copper upon human , 125I-human and rat Axcex21-40 aggregation into  greater than 0.2xcexc it particles. Stock human and rat Axcex21-40 peptide solutions (16 xcexcM) in water were pre-filtered (Spin-X, Costar, 0.2xcexc cellulose acetate, 700 g), brought to 100 mM NaCl, 20 mM Tris-HCl, pH 7.4 (buffer 1)xc2x1EDTA (50 xcexcM) or metal chloride salts, incubated (30 minutes, 37xc2x0 C.) and then filtered again (700 g, 4 minutes). The fraction of the Axcex21-40 in the filtrate was calculated by the ratio of the filtrate OD214 (the response of the OD214, titrated against human and rat Axcex21-40 concentrations (up to 20 xcexcM in the buffers used in these experiments), was determined to be linear) relative to the OD214 of the unfiltered sample. All data points are in triplicate, unless indicated. (FIG. 9) The graph shows the proportions of Axcex21-40 incubated xc2x1Zn2+ (25 xcexcM) or Cu2+ or EDTA (50 xcexcM) and then filtered through 0.2xcexc, titrated against peptide concentration.
FIG. 10. The amino acid sequence of human Axcex2 peptide SEQ ID NO:1. The amino acid sequence of human Axcex2 peptide is depicted and amino acid positions are numbered.